Abstract
Brachypodium distachyon is a promising model system for the structural and functional genomics of temperate grasses because of its physical, genetic and genome attributes. The sequencing of the inbred line Bd21 (http://www.brachypodium.org) started in 2007. However, a transformation method remains to be developed for the community standard line Bd21. In this article, a facile, efficient and rapid transformation system for Bd21 is described using Agrobacterium-mediated transformation of compact embryogenic calli (CEC) derived from immature embryos. Key features of this system include: (i) the use of the green fluorescent protein (GFP) associated with hygromycin selection for rapid identification of transgenic calli and plants; (ii) the desiccation of CEC after inoculation with Agrobacterium; (iii) the utilization of Bd21 plants regenerated from tissue culture as a source of immature embryos; (iv) the control of the duration of the selection process; and (v) the supplementation of culture media with CuSO4 prior to and during the regeneration of transgenic plants. Approximately 17% of CEC produced transgenic plants, enabling the generation of hundreds of T-DNA insertion lines per experiment. GFP expression was observed in primary transformed Bd21 plants (T0) and their progeny (T1). The Mendelian inheritance of the transgenes was confirmed. An adaptor-anchor strategy was developed for efficient retrieval of flanking sequence tags (FSTs) of T-DNA inserts, and the resulting sequences are available in public databases. The production of T-DNA insertion lines and the retrieval of associated FSTs reported here for the reference inbred line Bd21 will facilitate large-scale functional genomics research in this model system.
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Claims 5
Claim · model-written summary
The Bd21 transformation system yielded transgenic plants from approximately 17% of embryogenic calli.
- Taxon
- Brachypodium distachyon
Brachypodium distachyone1 - Material
- Bd21e2
- Outcome
- CEC produced transgenic plantse0
- Value
- Approximately 17%e0
Organisms pictured · Representative pictures of the matched species, not figures from the paper.
Verbatim
e0“Approximately 17% of CEC produced transgenic plants, enabling the generation of hundreds of T-DNA insertion lines per experiment”
e1“Brachypodium distachyon”
e2“transformation system for Bd21”
Claim · model-written summary
The system enabled hundreds of T-DNA insertion lines per experiment.
- Taxon
- Brachypodium distachyonBrachypodium distachyone1
- Outcome
- T-DNA insertion lines per experimente0
- Value
- hundredse0
Organisms pictured · Representative pictures of the matched species, not figures from the paper.
Verbatim
e0“enabling the generation of hundreds of T-DNA insertion lines per experiment”
e1“Brachypodium distachyon”
Claim · model-written summary
GFP expression occurred in primary transformed Bd21 plants and their progeny.
- Taxon
- Brachypodium distachyonBrachypodium distachyone1
- Material
- Bd21e0
- Gene
- GFPe0
- Outcome
- GFP expressione0
Organisms pictured · Representative pictures of the matched species, not figures from the paper.
Verbatim
e0“GFP expression was observed in primary transformed Bd21 plants (T0) and their progeny (T1)”
e1“Brachypodium distachyon”
Claim · model-written summary
Transgenes in transformed Bd21 plants showed Mendelian inheritance.
- Taxon
- Brachypodium distachyonBrachypodium distachyone1
- Material
- Bd21e2
- Outcome
- Mendelian inheritance of the transgenese0
Organisms pictured · Representative pictures of the matched species, not figures from the paper.
Verbatim
e0“The Mendelian inheritance of the transgenes was confirmed”
e1“Brachypodium distachyon”
e2“Bd21 plants regenerated from tissue culture”
Claim · model-written summary
An adaptor-anchor strategy retrieved flanking sequence tags of T-DNA inserts.
- Taxon
- Brachypodium distachyonBrachypodium distachyone1
- Outcome
- efficient retrieval of flanking sequence tags (FSTs) of T-DNA insertse0
Not reportedComparatorValue
Organisms pictured · Representative pictures of the matched species, not figures from the paper.
Verbatim
e0“An adaptor-anchor strategy was developed for efficient retrieval of flanking sequence tags (FSTs) of T-DNA inserts”
e1“Brachypodium distachyon”
e2“resulting sequences are available in public databases”